Project Summary Zika virus (ZIKV) was once an obscure mosquito-borne flavivirus that caused only sporadic human infections in Africa and Southeast Asia. Following a well-described outbreak on the Yap Islands in 2007, ZIKV spread across the islands of the Pacific and into the Western Hemisphere, emerging in Brazil in 2015 and spreading across South and Central America. Most significantly, ZIKV infection during pregnancy has been linked to microcephaly and severe birth defects. The Centers for Disease Control and Prevention (CDC) has issued testing algorithms for the diagnosis of infection in patients with suspected Zika fever that rely heavily on serological testing, particularly for pregnant women. Due to cross reaction with Dengue, virus-specific IgM detection, a critical component of the diagnostic arsenal for acute infection with mosquito-borne flaviviruses has been of limited utility during the current outbreak in the Americas, requiring time consuming and laborious plaque reduction neutralization testing (PRNT) for confirmation of the identity of the infecting virus. Our objective in this proposal is to create and validate a plasmonic gold (pGOLDTM) platform based screening test for IgG/IgM/IgA antibodies against a panel of flaviviral and alphaviral antigens, for highly specific diagnosis of ZIKV infection. The plasmonic gold platform is capable of amplifying near infrared (NIR) fluorescence by up to ~ 100 times, allowing analysis/quantification of multiple proteins or antibodies over 6-7 logs of dynamic range with high signal/noise. Antigen arrays were developed on plasmonic gold for multicolor, simultaneous detection of IgG/IgM/IgA antibody subtypes in type 1 diabetes and toxoplasmosis, with results matching highly sensitive methods performed in reference laboratories. pGOLD was the only demonstrated platform simultaneously detecting up to three antibody subtypes in a single assay using ~ 1 L serum sample volume. Two aims will be developed: First aim is to verify and validate a multiplexed serological detection of IgG, IgM and IgA against a panel of mosquito-borne viral antigens and investigate cross-reactivity. Second aim will be to a pGOLD nanotechnology-based multiplexed serologic assay for the specific diagnosis of ZIKV infection and differentiation from other flaviviruses. For these two aims, initial pilot testing highlights a multiplexed test of IgG and IgA antibodies afforded >90% sensitivity and specificity for differentiating ZIKV infected from DENV infected patients, with a positive ZIKV IgA level corresponding to a very recent ZIKV infection. These goals will definitively make the pGOLD accessible for universal screening of ZIKV infection with high specificity.